Various approaches have been described for performing assays to determine the presence or concentration of a specific microbial analyte, such as pathogenic microbial contamination of food. Frequently, a two-step process is followed wherein a first, rapid screening method is used to obtain initial assay results with negative results accepted as is. When a positive result is obtained, it is characterized as a “presumptive positive” result until it can be confirmed by the application of a second, alternative assay protocol applied to the sample. This process is followed because it is highly desirable, and in some cases required by government regulation, to corroborate a positive result in order to prevent the unnecessary recall and needless disposal of material implicated as containing a pathogen, such as food products.
In order for an alternative analysis protocol to be effective for the purpose of confirmation, it must meet several performance criteria. It should be selective, i.e. it should correctly identify a sample as not containing a target pathogenic microbe when in fact the microbe is not present. It should be sensitive, i.e. it should correctly identify a sample as containing a target pathogenic microbe when in fact the contaminant is present. It should have a low detection limit, i.e. it should give a positive result even when only a small number of target pathogen microbes are present.
Traditionally, these performance criteria have been met by applying cultural analysis protocols involving culture enrichment, selective and differential plating, and additional biochemical and serological methods. However, such cultural protocols suffer from the drawback that the time required to complete the analysis can easily extend over several days. This makes it difficult to acquire information in a timely manner upon which to base a decision regarding the recall or disposal of a perishable material implicated as potentially containing a pathogenic microbial contaminant by presumptive positive results obtained using a rapid screening method.
Thus, what is needed in the art is an alternative analysis method which meets the performance criteria necessary to corroborate presumptive positive results, and which can be completed rapidly enough to generate a result which can be acted upon in a timely manner.
Similarly, the present art of rapid screening suffers from the fact that either single signals are to be used to detect a given pathogen, in which case not all of the targets will be detected (they may not all have the target signal), or if multiple signals (multiplexes) are used to detect the pathogen/organism of interest, in matrixes such as food, water, environmental samples, and some clinical samples (such as urine and feces) where a diverse group of organisms are present, there is a distinct possibility that that the multiplex screen will detect a composite signal from more than one organism. For instance, if a food sample contains a non-toxigenic, eae negative E. coli O157, an enteropathogenic E. coli, and an E. coli, producing Shiga toxin, and the sample is screened with a multiplex PCR that targets eae, rfb, and stx, to detect pathogenic E. coli O157, then a composite signal consisting of three bands (eae, rfb, and stx) will be observed. In this instance, the data indicates that a pathogenic E. coli O157 is detected in the sample, but in reality, the toxin and the eae signal come from two other organisms. The presently available techniques will consider the sample a presumptive positive and it is then subjected to culture confirmation which will take 3-5 days.